Perturbation of the interactions of calmodulin with GRK5 using a natural product chemical probe.

G protein-coupled receptor (GPCR) kinases (GRKs) are responsible for initiating desensitization of activated GPCRs. GRK5 is potently inhibited by the calcium-sensing protein calmodulin (CaM), which leads to nuclear translocation of GRK5 and promotion of cardiac hypertrophy. Herein, we report the architecture of the Ca2+·CaM-GRK5 complex determined by small-angle X-ray scattering and negative-stain electron microscopy. Ca2+·CaM binds primarily to the small lobe of the kinase domain of GRK5 near elements critical for receptor interaction and membrane association, thereby inhibiting receptor phosphorylation while activating the kinase for phosphorylation of soluble substrates. To define the role of each lobe of Ca2+·CaM, we utilized the natural product malbrancheamide as a chemical probe to show that the C-terminal lobe of Ca2+·CaM regulates membrane binding while the N-terminal lobe regulates receptor phosphorylation and kinase domain activation. In cells, malbrancheamide attenuated GRK5 nuclear translocation and effectively blocked the hypertrophic response, demonstrating the utility of this natural product and its derivatives in probing Ca2+·CaM-dependent hypertrophy.

circDeep: deep learning approach for circular RNA classification from other long non-coding RNA.

MOTIVATION: Over the past two decades, a circular form of RNA (circular RNA), produced through alternative splicing, has become the focus of scientific studies due to its major role as a microRNA (miRNA) activity modulator and its association with various diseases including cancer. Therefore, the detection of circular RNAs is vital to understanding their biogenesis and purpose. Prediction of circular RNA can be achieved in three steps: distinguishing non-coding RNAs from protein coding gene transcripts, separating short and long non-coding RNAs and predicting circular RNAs from other long non-coding RNAs (lncRNAs). However, the available tools are less than 80 percent accurate for distinguishing circular RNAs from other lncRNAs due to difficulty of classification. Therefore, the availability of a more accurate and fast machine learning method for the identification of circular RNAs, which considers the specific features of circular RNA, is essential to the development of systematic annotation. RESULTS: Here we present an End-to-End deep learning framework, circDeep, to classify circular RNA from other lncRNA. circDeep fuses an RCM descriptor, ACNN-BLSTM sequence descriptor and a conservation descriptor into high level abstraction descriptors, where the shared representations across different modalities are integrated. The experiments show that circDeep is not only faster than existing tools but also performs at an unprecedented level of accuracy by achieving a 12 percent increase in accuracy over the other tools. AVAILABILITY AND IMPLEMENTATION: https://github.com/UofLBioinformatics/circDeep. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Taichunins E-T, Isopimarane Diterpenes and a 20-nor-Isopimarane, from Aspergillus taichungensis (IBT 19404): Structures and Inhibitory Effects on RANKL-Induced Formation of Multinuclear Osteoclasts.

Fifteen new isopimarane-type diterpenes, taichunins E-S (1-15), and a new 20-nor-isopimarane, taichunin T (16), together with four known compounds were isolated from Aspergillus taichungensis (IBT 19404). The structures of these new compounds were determined by NMR and mass spectroscopy, and their absolute configurations were analyzed by NOESY and TDDFT calculations of ECD spectra. Taichunins G, K, and N (3, 7, and 10) completely inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced formation of multinuclear osteoclasts in RAW264 cells at 5 µM, with 3 showing 92% inhibition at a concentration of 0.2 µM.

Molecular Basis for Spirocycle Formation in the Paraherquamide Biosynthetic Pathway.

The paraherquamides are potent anthelmintic natural products with complex heptacyclic scaffolds. One key feature of these molecules is the spiro-oxindole moiety that lends a strained three-dimensional architecture to these structures. The flavin monooxygenase PhqK was found to catalyze spirocycle formation through two parallel pathways in the biosynthesis of paraherquamides A and G. Two new paraherquamides (K and L) were isolated from a ΔphqK strain of Penicillium simplicissimum, and subsequent enzymatic reactions with these compounds generated two additional metabolites, paraherquamides M and N. Crystal structures of PhqK in complex with various substrates provided a foundation for mechanistic analyses and computational studies. While it is evident that PhqK can react with various substrates, reaction kinetics and molecular dynamics simulations indicated that the dioxepin-containing paraherquamide L is the favored substrate. Through this effort, we have elucidated a key step in the biosynthesis of the paraherquamides and provided a rationale for the selective spirocyclization of these powerful anthelmintic agents.

Flavin-Dependent Monooxygenases NotI and NotI' Mediate Spiro-Oxindole Formation in Biosynthesis of the Notoamides.

The fungal indole alkaloids are a unique class of complex molecules that have a characteristic bicyclo[2.2.2]diazaoctane ring and frequently contain a spiro-oxindole moiety. While various strains produce these compounds, an intriguing case involves the formation of individual antipodes by two unique species of fungi in the generation of the potent anticancer agents (+)- and (-)-notoamide A. NotI and NotI' have been characterized as flavin-dependent monooxygenases that catalyze epoxidation and semi-pinacol rearrangement to form the spiro-oxindole center within these molecules. This work elucidates a key step in the biosynthesis of the notoamides and provides an evolutionary hypothesis regarding a common ancestor for production of enantiopure notoamides.

Structural basis of the Cope rearrangement and cyclization in hapalindole biogenesis.

Hapalindole alkaloids are a structurally diverse class of cyanobacterial natural products defined by their varied polycyclic ring systems and diverse biological activities. These complex metabolites are generated from a common biosynthetic intermediate by the Stig cyclases in three mechanistic steps: a rare Cope rearrangement, 6-exo-trig cyclization, and electrophilic aromatic substitution. Here we report the structure of HpiC1, a Stig cyclase that catalyzes the formation of 12-epi-hapalindole U in vitro. The 1.5-Å structure revealed a dimeric assembly with two calcium ions per monomer and with the active sites located at the distal ends of the protein dimer. Mutational analysis and computational methods uncovered key residues for an acid-catalyzed [3,3]-sigmatropic rearrangement, as well as specific determinants that control the position of terminal electrophilic aromatic substitution, leading to a switch from hapalindole to fischerindole alkaloids.

Stereoselective Synthesis of Baulamycin A.

New structural classes of antibiotics are rare, structurally novel broad-spectrum antibiotics exceptionally so. The recently discovered baulamycins constitute a remarkable example of these highly prized compounds and, as such, have attracted considerable attention in the form of both synthetic efforts and biological studies. For the first time, we report a gram-scale preparation of the common carbon framework of the baulamycin family, as well as the total synthesis of its most potent member, baulamycin A. Our approach employs highly stereoselective, catalyst-controlled asymmetric conjugate additions to thioesters to set key stereocenters, as well as the first reported use of "dry ozonolysis" to reveal a masked carboxylic acid in the total synthesis of a natural product.

Irpexine, an Isoindolinone Alkaloid Produced by Coculture of Endophytic Fungi, Irpex lacteus and Phaeosphaeria oryzae.

A new isoindolinone alkaloid, irpexine (1), was isolated as a racemate, along with a known green pigment, hypoxyxylerone (2), from the coculture of two endophytic fungi, Irpex lacteus and Phaeosphaeria oryzae. Compound 1 was found to be a newly produced metabolite of I. lacteus in the coculture with P. oryzae. Although 2 was produced in a monoculture of I. lacteus, its production was markedly enhanced by the coculture.

Control of Stereoselectivity in Diverse Hapalindole Metabolites is Mediated by Cofactor-Induced Combinatorial Pairing of Stig Cyclases.

Stereospecific polycyclic core formation of hapalindoles and fischerindoles is controlled by Stig cyclases through a three-step cascade involving Cope rearrangement, 6-exo-trig cyclization, and a final electrophilic aromatic substitution. Reported here is a comprehensive study of all currently annotated Stig cyclases, revealing that these proteins can assemble into heteromeric complexes, induced by Ca2+ , to cooperatively control the stereochemistry of hapalindole natural products.

Decoding cyclase-dependent assembly of hapalindole and fischerindole alkaloids.

The formation of C-C bonds in an enantioselective fashion to create complex polycyclic scaffolds in the hapalindole- and fischerindole- type alkaloids from Stigonematales cyanobacteria represents a compelling and urgent challenge in adapting microbial biosynthesis as a catalytic platform in drug development. Here we determine the biochemical basis for tri- and tetracyclic core formation in these secondary metabolites, involving a new class of cyclases that catalyze a complex cyclization cascade.

Taichunins A-D, Norditerpenes from Aspergillus taichungensis (IBT 19404).

Four new norditerpenes, taichunins A-D (1-4), were isolated from the fungus Aspergillus taichungensis (IBT 19404). Compound 1 has a new carbon framework. The absolute configurations were determined by the calculated ECD spectral method. Compound 1 was cytotoxic against HeLa cells with an IC50 value of 4.5 µM, whereas 2-4 were nontoxic at 50 µM.

Structural and stereochemical diversity in prenylated indole alkaloids containing the bicyclo[2.2.2]diazaoctane ring system from marine and terrestrial fungi.

Covering: up to February 2017 Various fungi of the genera Aspergillus, Penicillium, and Malbranchea produce prenylated indole alkaloids possessing a bicyclo[2.2.2]diazaoctane ring system. After the discovery of distinct enantiomers of the natural alkaloids stephacidin A and notoamide B, from A. protuberus MF297-2 and A. amoenus NRRL 35660, another fungi, A. taichungensis, was found to produce their diastereomers, 6-epi-stephacidin A and versicolamide B, as major metabolites. Distinct enantiomers of stephacidin A and 6-epi-stephacidin A may be derived from a common precursor, notoamide S, by enzymes that form a bicyclo[2.2.2]diazaoctane core via a putative intramolecular hetero-Diels-Alder cycloaddition. This review provides our current understanding of the structural and stereochemical homologies and disparities of these alkaloids. Through the deployment of biomimetic syntheses, whole-genome sequencing, and biochemical studies, a unified biogenesis of both the dioxopiperazine and the monooxopiperazine families of prenylated indole alkaloids constituted of bicyclo[2.2.2]diazaoctane ring systems is presented.

Unveiling sequential late-stage methyltransferase reactions in the meleagrin/oxaline biosynthetic pathway.

Antimicrobial and anti-proliferative meleagrin and oxaline are roquefortine C-derived alkaloids produced by fungi of the genus Penicillium. Tandem O-methylations complete the biosynthesis of oxaline from glandicoline B through meleagrin. Currently, little is known about the role of these methylation patterns in the bioactivity profile of meleagrin and oxaline. To establish the structural and mechanistic basis of methylation in these pathways, crystal structures were determined for two late-stage methyltransferases in the oxaline and meleagrin gene clusters from Penicillium oxalicum and Penicillium chrysogenum. The homologous enzymes OxaG and RoqN were shown to catalyze penultimate hydroxylamine O-methylation to generate meleagrin in vitro. Crystal structures of these enzymes in the presence of methyl donor S-adenosylmethionine revealed an open active site, which lacks an apparent base indicating that catalysis is driven by proximity effects. OxaC was shown to methylate meleagrin to form oxaline in vitro, the terminal pathway product. Crystal structures of OxaC in a pseudo-Michaelis complex containing sinefungin and meleagrin, and in a product complex containing S-adenosyl-homocysteine and oxaline, reveal key active site residues with His313 serving as a base that is activated by Glu369. These data provide structural insights into the enzymatic methylation of these alkaloids that include a rare hydroxylamine oxygen acceptor, and can be used to guide future efforts towards selective derivatization and structural diversification and establishing the role of methylation in bioactivity.

Isolation of a new indoxyl alkaloid, Amoenamide B, from Aspergillus amoenus NRRL 35600: biosynthetic implications and correction of the structure of Speramide B.

A new prenylated indoxyl alkaloid, Amoenamide B (1), was isolated from Aspergillus amoenus NRRL 35600 along with Asperochramide A (2). Although many prenylated oxyindole alkaloids, containing bicyclo[2.2.2]diazaoctane cores, have been isolated from the fungus of the genera Aspergillus and Penicillium to date, 1 is the fourth compound with the indoxyl unit containing the cores. During the structure elucidation of 1, we found that the planar structure matched to that of Speramide A (3), isolated from A. ochraceus KM007, but the reported structure of 3 was incorrect and turned out to be that of Taichunamide H (4), recently isolated from A. versicolor HDN11-84.

Function and Structure of MalA/MalA', Iterative Halogenases for Late-Stage C-H Functionalization of Indole Alkaloids.

Malbrancheamide is a dichlorinated fungal indole alkaloid isolated from both Malbranchea aurantiaca and Malbranchea graminicola that belongs to a family of natural products containing a characteristic bicyclo[2.2.2]diazaoctane core. The introduction of chlorine atoms on the indole ring of malbrancheamide differentiates it from other members of this family and contributes significantly to its biological activity. In this study, we characterized the two flavin-dependent halogenases involved in the late-stage halogenation of malbrancheamide in two different fungal strains. MalA and MalA' catalyze the iterative dichlorination and monobromination of the free substrate premalbrancheamide as the final steps in the malbrancheamide biosynthetic pathway. Two unnatural bromo-chloro-malbrancheamide analogues were generated through MalA-mediated chemoenzymatic synthesis. Structural analysis and computational studies of MalA' in complex with three substrates revealed that the enzyme represents a new class of zinc-binding flavin-dependent halogenases and provides new insights into a potentially unique reaction mechanism.

Enantioselective inhibitory abilities of enantiomers of notoamides against RANKL-induced formation of multinuclear osteoclasts.

The marine-derived Aspergillus protuberus MF297-2 and the terrestrial A. amoenus NRRL 35600 produce enantiomeric prenylated indole alkaloids. Investigation of biological activities of the natural and synthetic derivatives revealed that (-)-enantiomers of notoamides A and B, 6-epi-notoamide T, and stephacidin A inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenic differentiation of murine RAW264 cells more strongly than their respective (+)-enantiomers. Among them, (-)-6-epi-notoamide T was the most potent inhibitor with an IC50 value of 1.7µM.

Isolation of amoenamide A and five antipodal prenylated alkaloids from Aspergillus amoenus NRRL 35600.

A new prenylated alkaloid, Amoenamide A (6), was isolated from the fungus Aspergillus amoenus NRRL 35600. Previously, 6 was postulated to be a precursor of Notoamide E4 (21) converted from Notoamide E (16), which was a key precursor of the prenylated indole alkaloids in the fungi of the genus Aspergillus. We previously succeeded in the isolation of two pairs of antipodes, Stephacidin A (1) and Notoamide B (2), from A. amoenus and A. protuberus MF297-2 and expected the presence of other antipodes in the culture of A. amoenus. We here report five new antipodes (7-11) along with a new metabolite (12), which was isolated as a natural compound for the first time, from A. amoenus.

Synthesis and Biochemical Evaluation of Biotinylated Conjugates of Largazole Analogues: Selective Class I Histone Deacetylase Inhibitors.

The synthesis of biotinylated conjugates of synthetic analogues of the potent and selective histone deacetylase (HDAC) inhibitor largazole is reported. The thiazole moiety of the parent compound's cap group was derivatized to allow the chemical conjugation to biotin. The derivatized largazole analogues were assayed across a panel of HDACs 1-9 and retained potent and selective inhibitory activity towards the class I HDAC isoforms. The biotinylated conjugate was further shown to pull down HDACs 1, 2, and 3.

OxaD: A Versatile Indolic Nitrone Synthase from the Marine-Derived Fungus Penicillium oxalicum F30.

Indole alkaloids are a diverse class of natural products known for their wide range of biological activities and complex chemical structures. Rarely observed in this class are indolic nitrones, such as avrainvillamide and waikialoid, which possess potent bioactivities. Herein the oxa gene cluster from the marine-derived fungus Penicillium oxalicum F30 is described along with the characterization of OxaD, a flavin-dependent oxidase that generates roquefortine L, a nitrone-bearing intermediate in the biosynthesis of oxaline. Nitrone functionality in roquefortine L was confirmed by spectroscopic methods and 1,3-dipolar cycloaddition with methyl acrylate. OxaD is a versatile biocatalyst that converts an array of semisynthetic roquefortine C derivatives bearing indoline systems to their respective nitrones. This work describes the first implementation of a nitrone synthase as a biocatalyst and establishes a novel platform for late-stage diversification of a range of complex natural products.

Stereoselective Total Synthesis of (-)-Renieramycin T.

A stereoselective total synthesis of (-)-Renieramycin T (1t) from a key tetrahydroisoquinoline intermediate previously utilized in our formal total synthesis of Ecteinascidin 743 is described. The synthesis features a concise approach for construction of the pentacyclic framework using a Pictet-Spengler cyclization of bromo-substituted carbinolamine 17, which obviates the regioselectivity problem of the Pictet-Spengler cyclization. The results of cytotoxicity studies are also presented.